Bispecific Tetravalent Antibodies and Methods of Making and Using Thereof

ABSTRACT

A bispecific tetravalent antibody comprising an IgG having a pair of heavy chains and a pair of light chains, and two scFv components being connected to either C or N terminals of the heavy or light chains. The bispecific tetravalent antibody may have a binding specificity for two different members of EGFR family.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is continuation-in-part of U.S. patent application Ser.No. 15/119,694, filed Aug. 17, 2016, which is a National Stage Entry ofPCT/US15/66951, filed Dec. 19, 2015, which claims priority over U.S.Provisional Application No. 62/095,348, filed Dec. 22, 2014, titled“BISPECIFIC ANTIBODIES,” the disclosure of which are hereby incorporatedby reference in their entirety.

SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is Sequence Listing_ST25_0003 PCT2.txt. The textfile is about 227 KB, was created on Dec. 18, 2015, and is beingsubmitted electronically via EFS-Web.

TECHNICAL FIELD

The present disclosure generally relates to the technical field ofantibody therapeutic agents, and more particularly relates to bispecifictetravalent antibodies against two different members of EGFR family.

BACKGROUND

Overexpression and/or deregulation of members of the ErbB/HER receptorfamily such as EGFR, HER2, HER3, HER4 have been shown to play animportant role in tumorigenesis in cancers. Mutation and amplificationof EGFR or HER2 produce aberrant growth signal which activatesdownstream signaling pathway contributing to tumorigenesis. Therapeuticantibodies and small-molecule inhibitors directed against EGFR and HER2have been approved for use in the treatment of cancer (Arteaga et al.,Nature Reviews Clinical Oncology 9 16-32, January 2012). Monoclonalantibodies against members of EGFR family such as EGFR and HER2, havedemonstrated good clinical responses in colon cancer (Price et al., TheLancet Oncology 15(6), Pages 569-579, May 2014), squamous cell carcinomaof head and neck (Cohen, Cancer Treatment Reviews 40 (2014) 567-577),breast and gastric cancers (Arteaga et al., Nature Reviews ClinicalOncology 9 16-32, January 2012). Several therapeutic anti-EGFRantibodies, including cetuximab, panitumumab and nimotuzumab areapproved therapeutics for several cancers including metastaticcolorectal cancer, head and neck squamous cell carcinoma and glioma(Price and Cohen, Curr Treat Options Oncol. 2012 March; 13(1):35-46;Bode et al., Expert Opin Biol Ther. 2012 December; 12(12):1649-59).Unfortunately, many tumors that initially respond to these therapeuticagents eventually progress due to an acquired resistance to the agents(Jackman et al. J Clin Oncol 2010; 28:357-60). Therefore, there exists aneed for better cancer therapeutics.

SUMMARY

The disclosure provides bispecific tetravalent antibodies. Thebispecific tetravalent antibodies may include an immunoglobulin G (IgG)moiety with two heavy chains and two light chains, and two scFv moietiesbeing covalently connected to either C or N terminals of the heavy orlight chains. The IgG moiety may have a binding specificity to a firstmember of EGFR family. The scFv moiety may have a binding specificity toa second member of the EGFR family. The IgG moiety and two scFv moietiesare covalently connected to be functional as a bispecific tetravalentantibody. The objectives and advantages of the disclosure will becomeapparent from the following detailed description of preferredembodiments thereof in connection with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments according to the present disclosure will now bedescribed with reference to the FIGs, in which like reference numeralsdenote like elements.

FIG. 1 is a diagram showing the domain structures of an example bivalentmonospecific immunoglobulin G (IgG) antibody.

FIG. 2 is a diagram showing the domain structure of an exampletetravalent bispecific antibody comprising an IgG moiety and two scFvmoieties in accordance with one embodiment of the present invention.

FIG. 3 shows the domain structure diagrams of example tetravalentbispecific antibodies 1X1, 1X2, 1X3, 1X4, 1X4.2, 1X5 and 1X6.

FIG. 4 shows the VH domain sequence comparison between SI-1X4 andSI-1X4.2 showing the 5 amino acid differences.

FIGS. 5 and 6 are graphs showing monomeric EGFR binding by BLI.

FIGS. 7, 8, and 9 are graphs showing bispecific ELI binding.

FIG. 10 is a graph showing dimeric EGFR ELISA.

FIG. 11 shows binding kinetics of SI-105.2 and SI-1X4.2 with monomericEGFR as analyzed by Octet.

FIG. 12 shows flow cytometric analysis of SI-1X antibodies binding toA431 cells.

FIG. 13 shows flow cytometric analysis of SI-1X antibodies binding toBxPC3 cells.

FIG. 14 shows flow cytometric analysis of SI-1X4.2 antibody binding toFadu cells.

FIG. 15 shows flow cytometric analysis of SI-1X4.2 antibody binding toA431 cells.

FIG. 16 shows effect of SI-1X antibodies on A431 cell proliferation.

FIG. 17 shows effect of SI-1X antibodies on A431 cell proliferation.

FIG. 18 shows effect of SI-1X antibodies on BxPC3 cell proliferation.

FIG. 19 shows effect of SI-1X antibodies on BxPC3 cell proliferation.

FIG. 20 shows effect of SI-1X4.2 antibodies on Fadu cell proliferation.

FIG. 21 shows effect of SI-1X4.2 antibodies on A431 cell proliferation.

FIG. 22 shows ADCC activity of SI-1X antibodies on Fadu cell.

FIG. 23 shows ADCC activity of SI-1X antibodies on NCI-H1975 cells.

FIG. 24 shows the thermal melting of SI-1X antibodies to demonstratetheir stability.

FIG. 25 shows the serum stability of SI-1X antibodies over 7 daysperiod.

FIG. 26 is a graph showing the results of EGFR coated ELISA for the PKstudy in rat.

FIG. 27 is a graph showing the results of HER3 coated ELISA for the PKstudy in rat.

FIG. 28 is a graph showing the results of sandwich ELISA for the PKstudy in rat.

FIG. 29 is a graph showing a plot of mean tumor volume vs days in themouse xenograft study.

FIG. 30 is a graph showing a plot relative body weight vs weeks in themouse xenograft study.

DETAILED DESCRIPTION

The present disclosure This disclosure provides bispecific tetravalentantibodies with superior therapeutic properties or efficacies over thecurrently known anti-EGFR antibodies. In one embodiment, the antibodiestarget two members of EGFR family including, without limitation, EFFRand HER3. The bispecific tetravalent antibodies may inhibit both EGFRand HER3 mediated signaling simultaneously therefore overcome resistancein EGFR inhibitor or monoclonal antibody treatment.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” in Throughout this specificationand claims, the word “comprise,” or variations such as “comprises” or“comprising,” will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers include plural referents unless the context clearlydictates otherwise.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen-binding or variable region of the intactantibody. Examples of antibody fragments include Fv, Fab, Fab′, F(ab′)2,Fab′-SH; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995));single-chain antibody molecules (e.g. scFv). While in the presentdescription, and throughout the specification, reference is made toantibodies and various properties of antibodies, the same disclosurealso applies to functional antibody fragments, e.g. dual action Fabfragments.

In one aspect, the bispecific tetravalent antibodies may include animmunoglobulin G (IgG) moiety with two heavy chains and two lightchains, and two scFv moieties being covalently connected to either C orN terminals of the heavy or light chains. The IgG moiety may have abinding specificity to a first member of EGFR family. The scFv moietymay have a binding specificity to a second member of the EGFR family.The IgG moiety may provide stability to the scFv moiety. The bispecifictetravalent antibody may block signalling for both AKT and MAPK/ERKpathways and may mediate antibody dependent cell-mediated cytotoxicity(ADCC) towards cells expressing either one or both antigens. In oneembodiment, the bispecific tetravalent antibody is capable of bindingboth antigens simultaneously. In some embodiments, the bispecifictetravalent antibody provides stronger tumour inhibition inproliferation assays in vitro and in vivo than the mono-specificantibody parental control or combination of the mono-specific antibodyparental controls.

In one embodiment, the disclosure provides a bispecific tetravalentantibody having two IgG1 heavy chains, two kappa light chains, and twosingle chain Fv (scFv) domains. The two IgG1 heavy chains and kappalight chains form an IgG moiety with a binding specificity to a firstmember of the EGFR family. The two scFv domains have a bindingspecificity to a second member of the EGFR family, and each scFv domainis connected to the C-terminus of either of the IgG1 heavy chains by aconnector with an amino acid sequence (gly-gly-gly-gly-ser)_(n), alsoknown as (G₄S)_(n), to provide a IgG1-connector connection. n is anintegral of at least 1. For example, n may be 2, 3, 4, 5, 6, 7, 8, 9,10, or 17. Each scFv domain has a structure order of N terminus-variableheavy-linker-variable light-C terminus. The linker may have an aminoacid sequence of (gly-gly-gly-gly-ser)_(m), also known as (G₄S)_(m). mmay be an integral of at least 2 or at least 3. For example, m may be 3,4, 5, 6, 11, or 12. In some embodiments, at least one or both of theIgG1 heavy chains are humanized or human. In some embodiments, at leastone or both of the kappa light chains are humanized or human.

The EGFR family members may include EGFR, HER2, HER3, a fragment or aderivative thereof. In some embodiments, the first member of the EGFRfamily may be EGFR, HER2, a fragment or a derivative thereof. In someembodiments, the second member of the EGFR family may be HER3, afragment or a derivative thereof. In one embodiment, the IgG moiety mayhave a binding specificity for HER3. In one embodiment, the scFv domainsmay have a binding specificity for EGFR. In one embodiment, the IgGmoiety may have a binding specificity for HER3, and the scFv domains mayhave a binding specificity for EGFR. In one embodiment, the IgG moietymay have a binding specificity for EGFR. In one embodiment, the scFvdomains may have a binding specificity for HER3. In one embodiment, theIgG moiety may a binding specificity for EGFR, and the scFv domains mayhave a binding specificity for HER3.

In some embodiments, the C terminus of one or both of the IgG1 heavychains misses an amino acid residue. For example, the lysine reside maybe deleted from the C terminus of the IgG1 chain before the connector isfused onto the C-terminus. The deletion of the lysine residue makes theIgG1-connector connection resistant to protease activity.

In some embodiments, one or both of the IgG1 heavy chains contain twomutations in the CH3 domain. For example, the two mutations may bereversion to the common residues in human CH3 domain.

In some embodiments, the IgG1 heavy chains may an amino acid sequencesof or with at least 95%, 98% or 99% similarity to SEQ ID NO 7, 15, 23,31, 39, 47, and 127. In some embodiments, the IgG1 heavy chain,connector, and scFv domain may have an amino acid sequence of or with atleast 95%, 98% or 99% similarity to SEQ ID NO 56, 66, 76, 86, 98, 108,118, and 136. In some embodiments, the kappa light chains may have anamino acid sequence of or with at least 95%, 98% or 99% similarity toSEQ ID NO 3, 11, 19, 27, 35, 43, 51, 61, 71, 81, 92, 103, 113, 123, and131. In some embodiments, the variable light chain may an amino acidsequence of or with at least 95%, 98% or 99% similarity to SEQ ID NO 4,12, 20, 28, 36, 44, 52, 62, 72, 82, 93, 104, 114, 124, and 132. In someembodiment, the variable heavy chain may have an amino acid sequence ofor with at least 95%, 98% or 99% similarity to SEQ ID NO 8, 16, 24, 32,40, 48, 57, 67, 77, 87, 99, 109, 119, 128, and 137.

In some embodiments, the IgG moiety has a binding specificity for HER3,and the scFv domains have a binding specificity for EGFR. In oneembodiment, the IgG1 heavy chain, connector, and scFv domain have anamino acid sequence of SEQ ID NO 56, and the kappa light chain has anamino acid sequence of SEQ ID NO 51. In one embodiment, the IgG1 heavychain, connector, and scFv domain have an amino acid sequence of SEQ IDNO 76, and the kappa light chain has an amino acid sequence of SEQ ID NO71. In one embodiment, the IgG1 heavy chain, connector, and scFv domainhave an amino acid sequence of SEQ ID NO 108, and the kappa light chainhas an amino acid sequence of SEQ ID NO 103.

In some embodiments, the IgG moiety has a binding specificity for EGFR,and the scFv domains have a binding specificity for HER3. In oneembodiment, the IgG1 heavy chain, connector, and scFv domain have anamino acid sequence of SEQ ID NO 66, and the kappa light chain has anamino acid sequence of SEQ ID NO 61. In one embodiment, the IgG1 heavychain, connector, and scFv domain have an amino acid sequence of SEQ IDNO 86, and the kappa light chain has an amino acid sequence of SEQ ID NO81. In one embodiment, the IgG1 heavy chain, connector, and scFv domainhave an amino acid sequence of SEQ ID NO 98, and the kappa light chainhas an amino acid sequence of SEQ ID NO 92. In one embodiment, the IgG1heavy chain, connector, and scFv domain have an amino acid sequence ofSEQ ID NO 118, and the kappa light chain has an amino acid sequence ofSEQ ID NO 113. In one embodiment, the IgG1 heavy chain, connector, andscFv domain have an amino acid sequence of SEQ ID NO 136, and the kappalight chain has an amino acid sequence of SEQ ID NO 131.

The bispecific tetravalent antibodies have the activity of inhibitingcancer cell growth. In certain embodiments, an antibody of the inventionhas a dissociation constant (Kd) of ≤80 nM, ≤50 nM, ≤30 nM, ≤20 nM, ≤10nM, or ≤0.1 nM for its target EGRF or HER3. The antibody may bind toboth targets simultaneously. In some embodiments, the antibody binds toEGRF and HER3 with a Kd less than 50 nM. In some embodiments, theantibody binds to EGRF and/or HER3 with a Kd less than 40, 30, 25, 20,19, 18 or 10 nM. In one embodiment, the antibody binds to EGRF with a Kdless than 30 nM and binds to HER3 with a Kd less than 30 nM. In oneembodiment, the antibody binds to EGRF with a Kd less than 50 nM andbinds to HER3 with a Kd less than 50 nM simultaneously.

In another aspect, the disclosure provides isolated nucleic acidsencoding the bispecific tetravalent antibodies or its sub-componentdisclosed herein. The sub-component may be the IgG1 heavy chain, thekappa light chain, the variable light chain, or the variable heavychain.

In a further aspect, the disclosure provides expression vectors havingthe isolated nucleic acids encoding the bispecific tetravalent antibodyor its sub-component disclosed herein. The vectors may be expressible ina host cell. The host cell may be prokaryotic or eukaryotic.

In a further aspect, the disclosure provides host cells having theisolated nucleic acids encoding the bispecific tetravalent antibodiesdisclosed herein or the expression vectors including such nucleic acidsequences.

In a further aspect, the disclosure provides methods for producingbispecific tetravalent antibodies. In one embodiment, the method mayinclude culturing the above-described host cells so that the antibody isproduced.

In a further aspect, the disclosure provides immunoconjugates includingthe bispecific tetravalent antibodies described herein and a cytotoxicagent.

In a further aspect, the disclosure provides pharmaceuticalcompositions. The pharmaceutical composition may include the bispecifictetravalent antibodies or the immunoconjugates described herein and apharmaceutically acceptable carrier. In some embodiments, thecomposition may further include radioisotope, radionuclide, a toxin, atherapeutic agent, a chemotherapeutic agent or a combination thereof.

In a further aspect, the disclosure provides methods of treating asubject with a cancer. In one embodiment, the method includes the stepof administering to the subject an effective amount of a bispecifictetravalent antibody described herein. The cancer may include cellsexpressing at least two members of EGFR family including, for example,EGFR, HER2, HER3, a fragment or a derivative thereof. The cancer may bebreast cancer, colorectal cancer, pancreatic cancer, head and neckcancer, melanoma, ovarian cancer, prostate cancer, and non-small lungcell cancer, glioma, esophageal cancer, nasopharyngeal cancer, analcancer, rectal cancer, gastric cancer, bladder cancer, cervical cancerand brain cancer.

In one embodiment, the method may further include co-administering aneffective amount of a therapeutic agent. The therapeutic agent may be,for example, an antibody, a chemotherapy agent, a cytotoxic agent, anenzyme, or a combination thereof. In some embodiments, the therapeuticagent may be an anti-estrogen agent, a receptor tyrosine inhibitor, or acombination thereof. In some embodiments, the therapeutic agent may bebiologics. In one embodiment, the therapeutic agent may be a checkpointinhibitor. In some embodiments, the therapeutic agent may include PD1,PDL1, CTLA4, 4-1BB, OX40, GITR, TIM3, LAG3, TIGIT, CD40, CD27, HVEM,BTLA, VISTA, B7H4, a derivative, a conjugate, or a fragment thereof. Insome embodiments, the therapeutic agent may be capecitabine, cisplatin,trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole,aminoglutethimide, testolactone, vorozole, formestane, fadrozole,letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib,pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, nab-palitaxel, ora derivative thereof. In some embodiments, the subject in need of suchtreatment is a human.

In one embodiment, the disclosure provides methods for treating asubject by administering to the subject an effective amount of thebispecific tetravalent antibody to inhibit a biological activity of aHER receptor.

In one embodiment, the disclosure provides solutions having an effectiveconcentration of the bispecific tetravalent antibody. In one embodiment,the solution is blood plasma in a subject.

A diagram of the general structure of IgG is shown in FIG. 1.

A diagram of the representative structure of the bispecific tetravalentantibodies according to some embodiments is shown in FIG. 2. In thisexample, the bispecific tetravalent antibody includes two human IgG1heavy chains, two human kappa light chains, and two single chain Fv(scFv) domains. The two human IgG1 heavy chains and human kappa lightchains form an IgG moiety with a binding specificity to one member ofthe EGFR family, and each of the two scFv domains is connected to theC-terminal residue of either of the human IgG1 heavy chains by aconnector with an amino acid sequence ofgly-gly-gly-gly-ser-gly-gly-gly-gly-ser ((G₄S)₂). Each scFv domain is inthe order: N terminus-variable heavy-linker-variable light-C terminus.The linker is comprised of amino acid sequence ofgly-gly-gly-gly-ser-gly-gly-gly-gly-ser-gly-gly-gly-gly-ser, also knownas (G₄S)₃. For some embodiments of the bispecific tetravalentantibodies, the CH1, CH2, CH3, CL, Connector and Linker amino acidsequences are identical. Each bispecific tetravalent antibody has abivalent anti-HER3 binding specificity on one end of the antibody and abivalent anti-EGFR binding specificity on the other end. One pair ofanti-HER3 variable heavy chain and variable light chain is designated as1C1, and four pairs of anti-EGFR variable heavy chains and variablelight chains are designated as 1C3, 1C5, 1C5.2, 1C6 and 1C6.4,respectively. The bispecific tetravalent antibodies are designated as1X1, 1X2, 1X3, 1X4, 1X4.2, 1X5, 1X5.2, 1X6, and 1X6.4.

In addition, a control molecule 1C4 (also designated as SI-1C4) was usedin some of the studies. 1C4 is a bispecific antibody against EGFR andHER3 built on the two-in-one platform described by Schaefer et. al.,2011 (Schaefer et al., Cancer Cell. 2011 Oct. 18; 20(4):472-86). IC4 hasa similar structure to a monoclonal antibody. The molecule can bind toeither EGFR or HER3 on each Fab arm, but cannot engage both targetssimultaneously on each Fab arm.

Variable light chain, variable heavy chain and single chain Fv (scFv)DNA fragments were generated by gene synthesis through an outsidevendor. Human Gamma-1 heavy chain and human kappa light chain DNAfragments were generated by gene synthesis through an outside vendor.The fragments were assembled together by DNA ligation using restrictionsites and cloned into a vector that is designed for transient expressionin mammalian cells. The vector contains a strong CMV-derived promoter,and other upstream and downstream elements required for transientexpression. The resulting IgG expression plasmids were verified ascontaining the expected DNA sequences by DNA sequencing.

Transient expression of the antibody constructs was achieved usingtransfection of suspension-adapted HEK293F cells with linear PEI asdescribed elsewhere (see CSH Protocols; 2008; doi:10.1101/pdb.prot4977).Antibodies were purified from the resulting transfection supernatantsusing protein affinity chromatography and size exclusion chromatographyif needed. Protein quality is analysed by Superdex 200 column. Proteinused for all the assays have a purity of greater than 90%.

The bispecific antibody may be used for the treatment of cancer typeswith EGFR and HER3 co-expressions, including without limitation coloncancer, head and neck squamous cell carcinoma, lung cancer, glioma,pancreatic cancer, nasopharyngeal cancer and other cancer types.

The bispecific antibody is of tetravalent dual specificity. The exampleantibody may include an IgG and two scFv, which provides two differentbinding specificities compared to mono-specific antibody IgG. The IgGcomponent provides stability and improved serum half-life over otherbispecific antibodies that used only scFv such as BiTE technology(Lutterbuese et al., Proceedings of the National Academy of Sciences ofthe United States of America 107.28 (2010): 12605-12610. PMC. Web. 2Dec. 2014) and others (for example, U.S. Pat. No. 7,332,585B2). It isalso capable of mediating ADCC while those without Fc component cannot(for example, U.S. Pat. No. 7,332,585B2). The tetravalent dualspecificity nature provides the bispecific antibody a simultaneousbinding capability over some other bispecific antibodies, which may onlybind one antigen at a time (Schanzer et al, Antimicrob. AgentsChemother. 2011, 55(5):2369; EP272942A1).

For the convenient of narration, the sequences of or related to thebispecific antibodies are summarized in TABLE 1 herein below.

TABLE 1 Summary of nucleotide and amino acid sequences of or related tothe bispecific antibodies. SI-1C1 SEQUENCES SEQ ID NO 1 SI-1C1 LIGHTCHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 2 SI-1C1 LIGHT CHAINVARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 3 SI-1C1 LIGHT CHAINFULL-LENGTH AMINO ACID SEQUENCE. HUMAN KAPPA CONSTANT DOMAIN ISUNDERLINED SEQ ID NO 4 SI-1C1 LIGHT CHAIN VARIABLE LIGHT CHAIN AMINOACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SEQ IDNO 5 SI-1C1 HEAVY CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 6SI-1C1 HEAVY CHAIN VARIABLE HEAVY CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 7SI-1C1 HEAVY CHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMAN GAMMA-1 DOMAINIS UNDERLINED SEQ ID NO 8 SI-1C1 HEAVY CHAIN VARIABLE HEAVY CHAIN AMINOACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SI-1C3SEQUENCES SEQ ID NO 9 SI-1C3 LIGHT CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCESEQ ID NO 10 SI-1C3 LIGHT CHAIN VARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCESEQ ID NO 11 SI-1C3 LIGHT CHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMANKAPPA CONSTANT DOMAIN IS UNDERLINED SEQ ID NO 12 SI-1C3 LIGHT CHAINVARIABLE LIGHT CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITY DETERMININGREGIONS ARE UNDERLINED SEQ ID NO 13 SI-1C3 HEAVY CHAIN FULL-LENGTHNUCLEOTIDE SEQUENCE SEQ ID NO 14 SI-1C3 HEAVY CHAIN VARIABLE HEAVY CHAINNUCLEOTIDE SEQUENCE SEQ ID NO 15 SI-1C3 HEAVY CHAIN FULL-LENGTH AMINOACID SEQUENCE. HUMAN GAMMA-1 DOMAIN IS UNDERLINED SEQ ID NO 16 SI-1C3HEAVY CHAIN VARIABLE HEAVY CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITYDETERMINING REGIONS ARE UNDERLINED SI-1C4 SEQUENCES SEQ ID NO 17 SI-1C4LIGHT CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 18 SI-1C4 LIGHTCHAIN VARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 19 SI-1C4 LIGHTCHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMAN KAPPA CONSTANT DOMAIN ISUNDERLINED SEQ ID NO 20 SI-1C4 LIGHT CHAIN VARIABLE LIGHT CHAIN AMINOACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SEQ IDNO 21 SI-1C4 HEAVY CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 22SI-1C4 HEAVY CHAIN VARIABLE HEAVY CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 23SI-1C4 HEAVY CHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMAN GAMMA-1 DOMAINIS UNDERLINED SEQ ID NO 24 SI-1C4 HEAVY CHAIN VARIABLE HEAVY CHAIN AMINOACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SI-1C5SEQUENCES SEQ ID NO 25 SI-1C5 LIGHT CHAIN FULL-LENGTH NUCLEOTIDESEQUENCE SEQ ID NO 26 SI-1C5 LIGHT CHAIN VARIABLE LIGHT CHAIN NUCLEOTIDESEQUENCE SEQ ID NO 27 SI-1C5 LIGHT CHAIN FULL-LENGTH AMINO ACIDSEQUENCE. HUMAN KAPPA CONSTANT DOMAIN IS UNDERLINED SEQ ID NO 28 SI-1C5LIGHT CHAIN VARIABLE LIGHT CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITYDETERMINING REGIONS ARE UNDERLINED SEQ ID NO 29 SI-1C5 HEAVY CHAINFULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 30 SI-1C5 HEAVY CHAIN VARIABLEHEAVY CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 31 SI-1C5 HEAVY CHAINFULL-LENGTH AMINO ACID SEQUENCE. HUMAN GAMMA-1 DOMAIN IS UNDERLINED SEQID NO 32 SI-1C5 HEAVY CHAIN VARIABLE HEAVY CHAIN AMINO ACID SEQUENCE.COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SI-1C5.2 SEQUENCESSEQ ID NO 33 SI1C5.2 LIGHT CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ IDNO 34 SI-1C5.2 LIGHT CHAIN VARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCE SEQID NO 35 SI-1C5.2 LIGHT CHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMANKAPPA CONSTANT DOMAIN IS UNDERLINED SEQ ID NO 36 SI-1C5.2 LIGHT CHAINVARIABLE LIGHT CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITY DETERMININGREGIONS ARE UNDERLINED SEQ ID NO 37 SI-1C5.2 HEAVY CHAIN FULL-LENGTHNUCLEOTIDE SEQUENCE SEQ ID NO 38 SI-1C5.2 HEAVY CHAIN VARIABLE HEAVYCHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 39 SI-1C5.2 HEAVY CHAIN FULL-LENGTHAMINO ACID SEQUENCE. HUMAN GAMMA-1 DOMAIN IS UNDERLINED SEQ ID NO 40SI-1C5.2 HEAVY CHAIN VARIABLE HEAVY CHAIN AMINO ACID SEQUENCE.COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SI-1C6 SEQUENCES SEQID NO 41 SI-1C6 LIGHT CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 42SI-1C6 LIGHT CHAIN VARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 43SI-1C6 LIGHT CHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMAN KAPPA CONSTANTDOMAIN IS UNDERLINED SEQ ID NO 44 SI-1C6 LIGHT CHAIN VARIABLE LIGHTCHAIN AMINO ACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS AREUNDERLINED SEQ ID NO 45 SI-1C6 HEAVY CHAIN FULL-LENGTH NUCLEOTIDESEQUENCE SEQ ID NO 46 SI-1C6 HEAVY CHAIN VARIABLE HEAVY CHAIN NUCLEOTIDESEQUENCE SEQ ID NO 47 SI-1C6 HEAVY CHAIN FULL-LENGTH AMINO ACIDSEQUENCE. HUMAN GAMMA-1 DOMAIN IS UNDERLINED SEQ ID NO 48 SI-1C6 HEAVYCHAIN VARIABLE HEAVY CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITYDETERMINING REGIONS ARE UNDERLINED SI-1X1 SEQUENCES SEQ ID NO 49 SI1X1LIGHT CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 50 SI-1X1 LIGHTCHAIN VARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO Si SI-1X1 LIGHTCHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMAN KAPPA CONSTANT DOMAIN ISUNDERLINED SEQ ID NO 52 SI-1X1 LIGHT CHAIN VARIABLE LIGHT CHAIN AMINOACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SEQ IDNO 53 SI1X1 BISPECIFIC HEAVY CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQID NO 54 SI-1X1 BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAIN NUCLEOTIDESEQUENCE SEQ ID NO 55 SI-1X1 BISPECIFIC HEAVY CHAIN SCFV NUCLEOTIDESEQUENCE SEQ ID NO 56 SI-1X1 BISPECIFIC HEAVY CHAIN FULL-LENGTH AMINOACID SEQUENCE. HUMAN GAMMA-1 DOMAIN IS UNDERLINED, CONNECTOR IS INITALICS, SCFV IS IN BOLD SEQ ID NO 57 SI-1X1 BISPECIFIC HEAVY CHAINVARIABLE HEAVY CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITY DETERMININGREGIONS ARE UNDERLINED SEQ ID NO 58 SI1X1 BISPECIFIC HEAVY CHAIN SCFVAMINO ACID SEQUENCE. ORDER:VH-LINKER-VL. COMPLEMENTARITY DETERMININGREGIONS ARE UNDERLINED. LINKER IS IN BOLD ITALICS SI-1X2 SEQUENCES SEQID NO 59 SI-1X2 LIGHT CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 60SI-1X2 LIGHT CHAIN VARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 61SI-1X2 LIGHT CHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMAN KAPPA CONSTANTDOMAIN IS UNDERLINED SEQ ID NO 62 SI-1X2 LIGHT CHAIN VARIABLE LIGHTCHAIN AMINO ACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS AREUNDERLINED SEQ ID NO 63 SI-1X2 BISPECIFIC HEAVY CHAIN FULL-LENGTHNUCLEOTIDE SEQUENCE SEQ ID NO 64 SI-1X2 BISPECIFIC HEAVY CHAIN VARIABLEHEAVY CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 65 SI-1X2 BISPECIFIC HEAVYCHAIN SCFV NUCLEOTIDE SEQUENCE SEQ ID NO 66 SI-1X2 BISPECIFIC HEAVYCHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMAN GAMMA-1 DOMAIN ISUNDERLINED, CONNECTOR IS IN ITALICS, SCFV IS IN BOLD SEQ ID NO 67 SI-1X2BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAIN AMINO ACID SEQUENCE.COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SEQ ID NO 68 SI-1X2BISPECIFIC HEAVY CHAIN SCFV AMINO ACID SEQUENCE. ORDER:VH-LINKER-VL.COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED. LINKER IS IN BOLDITALICS SI-1X3 SEQUENCES SEQ ID NO 69 SI-1X3 LIGHT CHAIN FULL-LENGTHNUCLEOTIDE SEQUENCE SEQ ID NO 70 SI-1X3 LIGHT CHAIN VARIABLE LIGHT CHAINNUCLEOTIDE SEQUENCE SEQ ID NO 71 SI-1X3 LIGHT CHAIN FULL-LENGTH AMINOACID SEQUENCE. HUMAN KAPPA CONSTANT DOMAIN IS UNDERLINED SEQ ID NO 72SI-1X3 LIGHT CHAIN VARIABLE LIGHT CHAIN AMINOACID SEQUENCE.COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SEQ ID NO 73 SI-1X3BISPECIFIC HEAVY CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 74SI-1X3 BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAIN NUCLEOTIDE SEQUENCESEQ ID NO 75 SI-1X3 BISPECIFIC HEAVY CHAIN SCFV NUCLEOTIDE SEQUENCE SEQID NO 76 SI-1X3 BISPECIFIC HEAVY CHAIN FULL-LENGTH AMINO ACID SEQUENCE.HUMAN GAMMA-1 DOMAIN IS UNDERLINED, CONNECTOR IS IN ITALICS, SCFV IS INBOLD SEQ ID NO 77 SI-1X3 BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAINAMINO ACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINEDSEQ ID NO 78 SI-1X3 BISPECIFIC HEAVY CHAIN SCFV AMINO ACID SEQUENCE.ORDER:VH-LINKER-VL. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED.LINKER IS IN BOLD ITALICS SI-1X4 SEQUENCES SEQ ID NO 79 SI-1X4 LIGHTCHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 80 SI-1X4 LIGHT CHAINVARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 81 SI-1X4 LIGHT CHAINFULL-LENGTH AMINO ACID SEQUENCE. HUMAN KAPPA CONSTANT DOMAIN ISUNDERLINED SEQ ID NO 82 SI-1X4 LIGHT CHAIN VARIABLE LIGHT CHAIN AMINOACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SEQ IDNO 83 SI-1X4 BISPECIFIC HEAVY CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQID NO 84 SI-1X4 BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAIN NUCLEOTIDESEQUENCE SEQ ID NO 85 SI-1X4 BISPECIFIC HEAVY CHAIN SCFV NUCLEOTIDESEQUENCE SEQ ID NO 86 SI-1X4 BISPECIFIC HEAVY CHAIN FULL-LENGTH AMINOACID SEQUENCE. HUMAN GAMMA-1 DOMAIN IS UNDERLINED, CONNECTOR IS INITALICS, SCFV IS IN BOLD SEQ ID NO 87 SI-1X4 BISPECIFIC HEAVY CHAINVARIABLE HEAVY CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITY DETERMININGREGIONS ARE UNDERLINED SEQ ID NO 88 SI-1X4 BISPECIFIC HEAVY CHAIN SCFVAMINO ACID SEQUENCE. ORDER:VH-LINKER-VL. COMPLEMENTARITY DETERMININGREGIONS ARE UNDERLINED. LINKER IS IN BOLD ITALICS SI-1X4.2 SEQUENCES SEQID NO 89 SI-1X4.2 LIGHT CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO90 SI-1X4.2 LIGHT CHAIN VARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCE SEQ IDNO 91 SI-1X4.2 LIGHT CHAIN VARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCECODON OPTIMIZED FOR CHO EXPRESSION SEQ ID NO 92 SI-1X4.2 LIGHT CHAINFULL-LENGTH AMINO ACID SEQUENCE. HUMAN KAPPA CONSTANT DOMAIN ISUNDERLINED SEQ ID NO 93 SI-1X4.2 LIGHT CHAIN VARIABLE LIGHT CHAIN AMINOACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SEQ IDNO 94 SI-1X4.2 BISPECIFIC HEAVY CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCESEQ ID NO 95 SI-1X4.2 BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAINNUCLEOTIDE SEQUENCE SEQ ID NO 96 SI-1X4.2 BISPECIFIC HEAVY CHAINVARIABLE HEAVY CHAIN NUCLEOTIDE SEQUENCE CODON OPTIMIZED FOR CHOEXPRESSION SEQ ID NO 97 SI-1X4.2 BISPECIFIC HEAVY CHAIN SCFV NUCLEOTIDESEQUENCE SEQ ID NO 98 SI-1X4.2 BISPECIFIC HEAVY CHAIN FULL-LENGTH AMINOACID SEQUENCE. HUMAN GAMMA-1 DOMAIN IS UNDERLINED, CONNECTOR IS INITALICS, SCFV IS IN BOLD SEQ ID NO 99 SI-1X4.2 BISPECIFIC HEAVY CHAINVARIABLE HEAVY CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITY DETERMININGREGIONS ARE UNDERLINED SEQ ID NO 100 SI-1X4.2 BISPECIFIC HEAVY CHAINSCFV AMINO ACID SEQUENCE. ORDER:VH-LINKER-VL. COMPLEMENTARITYDETERMINING REGIONS ARE UNDERLINED. LINKER IS IN BOLD ITALICS SI-1X5SEQUENCES SEQ ID NO 101 SI-1X5 LIGHT CHAIN FULL-LENGTH NUCLEOTIDESEQUENCE SEQ ID NO 102 SI-1X5 LIGHT CHAIN VARIABLE LIGHT CHAINNUCLEOTIDE SEQUENCE SEQ ID NO 103 SI-1X5 LIGHT CHAIN FULL-LENGTH AMINOACID SEQUENCE. HUMAN KAPPA CONSTANT DOMAIN IS UNDERLINED SEQ ID NO 104SI-1X5 LIGHT CHAIN VARIABLE LIGHT CHAIN AMINO ACID SEQUENCE.COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SEQ ID NO 105 SI-1X5BISPECIFIC HEAVY CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 106SI-1X5 BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAIN NUCLEOTIDE SEQUENCESEQ ID NO 107 SI-1X5 BISPECIFIC HEAVY CHAIN SCFV NUCLEOTIDE SEQUENCE SEQID NO 108 SI-1X5 BISPECIFIC HEAVY CHAIN FULL-LENGTH AMINO ACID SEQUENCE.HUMAN GAMMA-1 DOMAIN IS UNDERLINED, CONNECTOR IS IN ITALICS, SCFV IS INBOLD SEQ ID NO 109 SI-1X5 BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAINAMINO ACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINEDSEQ ID NO 110 SI-1X5 BISPECIFIC HEAVY CHAIN SCFV AMINO ACID SEQUENCE.ORDER:VH-LINKER-VL. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED.LINKER IS IN BOLD ITALICS SI-1X6 SEQUENCES SEQ ID NO 111 SI-1X6 LIGHTCHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 112 SI-1X6 LIGHT CHAINVARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 113 SI-1X6 LIGHTCHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMAN KAPPA CONSTANT DOMAIN ISUNDERLINED SEQ ID NO 114 SI-1X6 LIGHT CHAIN VARIABLE LIGHT CHAIN AMINOACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SEQ IDNO 115 SI-1X6 BISPECIFIC HEAVY CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQID NO 116 SI-1X6 BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAIN NUCLEOTIDESEQUENCE SEQ ID NO 117 SI-1X6 BISPECIFIC HEAVY CHAIN SCFV NUCLEOTIDESEQUENCE SEQ ID NO 118 SI-1X6 BISPECIFIC HEAVY CHAIN FULL-LENGTH AMINOACID SEQUENCE. HUMAN GAMMA-1 DOMAIN IS UNDERLINED, CONNECTOR IS INITALICS, SCFV IS IN BOLD SEQ ID NO 119 SI-1X6 BISPECIFIC HEAVY CHAINVARIABLE HEAVY CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITY DETERMININGREGIONS ARE UNDERLINED SEQ ID NO 120 SI-1X6 BISPECIFIC HEAVY CHAIN SCFVAMINO ACID SEQUENCE. ORDER:VH-LINKER-VL. COMPLEMENTARITY DETERMININGREGIONS ARE UNDERLINED. LINKER IS IN BOLD ITALICS SI-1C6.2 SEQUENCES SEQID NO 121 SI-1C6.2 LIGHT CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO122 SI-1C6.2 LIGHT CHAIN VARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCE SEQ IDNO 123 SI-1C6.2 LIGHT CHAIN FULL-LENGTH AMINO ACID SEQUENCE. HUMAN KAPPACONSTANT DOMAIN IS UNDERLINED SEQ ID NO 124 SI-1C6.2 LIGHT CHAINVARIABLE LIGHT CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITY DETERMININGREGIONS ARE UNDERLINED SEQ ID NO 125 SI-1C6.2 HEAVY CHAIN FULL-LENGTHNUCLEOTIDE SEQUENCE SEQ ID NO 126 SI-1C6.2 HEAVY CHAIN VARIABLE HEAVYCHAIN NUCLEOTIDE SEQUENCE SEQ ID NO 127 SI-1C6.2 HEAVY CHAIN FULL-LENGTHAMINO ACID SEQUENCE. HUMAN GAMMA-1 DOMAIN IS UNDERLINED SEQ ID NO 128SI-1C6.2 HEAVY CHAIN VARIABLE HEAVY CHAIN AMINO ACID SEQUENCE.COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED SI-1X6.4 SEQUENCESSEQ ID NO 129 SI-1X6.4 LIGHT CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQID NO 130 SI-1X6.4 LIGHT CHAIN VARIABLE LIGHT CHAIN NUCLEOTIDE SEQUENCESEQ ID NO 131 SI-1X6.4 LIGHT CHAIN FULL-LENGTH AMINO ACID SEQUENCE.HUMAN KAPPA CONSTANT DOMAIN IS UNDERLINED SEQ ID NO 132 SI-1X6.4 LIGHTCHAIN VARIABLE LIGHT CHAIN AMINO ACID SEQUENCE. COMPLEMENTARITYDETERMINING REGIONS ARE UNDERLINED SEQ ID NO 133 SI-1X6.4 BISPECIFICHEAVY CHAIN FULL-LENGTH NUCLEOTIDE SEQUENCE SEQ ID NO 134 SI-1X6.4BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAIN NUCLEOTIDE SEQUENCE SEQ IDNO 135 SI-1X6.4 BISPECIFIC HEAVY CHAIN SCFV NUCLEOTIDE SEQUENCE SEQ IDNO 136 SI-1X6.4 BISPECIFIC HEAVY CHAIN FULL-LENGTH AMINO ACID SEQUENCE.HUMAN GAMMA-1 DOMAIN IS UNDERLINED, CONNECTOR IS IN ITALICS, SCFV IS INBOLD SEQ ID NO 137 SI-1X6.4 BISPECIFIC HEAVY CHAIN VARIABLE HEAVY CHAINAMINO ACID SEQUENCE. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINEDSEQ ID NO 138 SI-1X6.4 BISPECIFIC HEAVY CHAIN SCFV AMINO ACID SEQUENCE.ORDER:VH-LINKER-VL. COMPLEMENTARITY DETERMINING REGIONS ARE UNDERLINED.LINKER IS IN BOLD ITALICS

EXAMPLES

While The following examples are provided by way of illustration onlyand not by way of limitation. Those of skill in the art will readilyrecognize a variety of non-critical parameters that could be changed ormodified to yield essentially the same or similar results.

Example 1: Sequence Differences Between SI-1X4 and SI-1X4.2

SI-1X4.2 is a modification of SI-1X4 molecule and contained 5 amino acidchanges as follows: V71A, T75S, N76S, A93T and S107T using the Kabatnumbering system. Some of these changes especially positions 75, 76 and93 potentially made interaction with antigen even though these are notin the CDR loops and are essential for binding and activity. FIG. 4shows the 5 amino acid differences between SI-1X4.2 and SI-1X4.

Example 2: Characterization of Antibodies Against Epidermal GrowthFactor Receptor Using BLI

Monomeric EGFR extracellular domain binding was measured in a biolayerinterferometry (BLI) binding assay on a BLItz instrument (ForteBio,Inc.). 25 μg/mL of SI-1C3, SI-1C4, SI-1C6, SI-1X1, SI-1X2, SI-1X5, andSI-1X6 were diluted in PBS and captured on anti-hulgG Fc BLItz biosensortips for 120 seconds. Tips were washed for 30 seconds in PBS and movedto an EGFR (ProSpec Bio, PKA-344) sample for binding at 588 nM. Bindingof EGFR ECD to the tips was recorded as biolayer interferometry signals(Δnm) over an association time of 120 seconds. Tips were moved to PBSand dissociation was observed for 240 seconds (*SI-1C6 dissociation timeof only 120 seconds observed). FIGS. 5 and 6 report data starting at theassociation step of EGFR to the antibody-loaded biosensor. Each Figureshows comparison to SI-1C4 as a benchmark antibody.

Since SI-1C3 and SI-1X2 share their EGFR binding domain displayed as aFab, their binding profiles are similar and stronger than the scFv formdisplayed on SI-1X1 (FIG. 6). Each has a very slow off-rate to EGFRcompared to SI-1C4 and is not affected by their on-rate. SI-1X1 may showweaker on-rate binding to EGFR, but stays bound very strongly. The sametrend is observed in FIG. 5, where the Fab versions of the EGFR bindingdomains displayed on SI-1C6 and SI-1X6 bind at a faster rate than theirrepresentative scFv displayed on SI-1X5. Having the EGFR binding domainon the Fab side of the bispecifics antibody appears to bind with fasteron-rates than the scFv versions, yet exhibit similar off-rates. SI-1X3and SI-1X4 do not exhibit monomeric EGFR binding in this assay (data notshown) and dimeric EGFR binding is investigated in an ELISA below.

Example 3: Characterization of Antibodies Against EGFR and Her3 UsingBL1

Bispecific binding to EGFR and Her3 extracellular domains was measuredin a biolayer interferometry (BLI) binding assay on a BLItz instrument(ForteBio, Inc.). 200 nM of SI-1C1, SI-1C3, SI-1C4, SI-1C6, SI-1X1,SI-1X2, SI-1X3, SI-1X4, SI-1X5, and SI-1X6 were diluted in 1X KineticsBuffer (ForteBio, Inc.) and captured on anti-hulgG Fc BLItz biosensortips for 120 seconds. Tips were washed in KB for 30 seconds and moved toan EGFR sample (ProSpec Bio, PKA-344) for binding at 200 nM. Binding ofEGFR ECD to the tips was recorded as biolayer interferometry signals(Δnm) over an association time of 120 seconds. Tips were moved to KB anddissociation was observed for 60 seconds. The process was repeated withHer3 ECD sample (Sino Biological, 10201-H08H-10) at 200 nM for 120seconds and a similar dissociation step of 60 seconds in KB. FIGS. 8-107-9 report data starting at the association step of EGFR to theantibody-loaded biosensor. Antibodies are able to exhibit simultaneousbispecific binding of EGFR and Her3 while being bound by the Fc to thesensor. As observed in FIG. 7 and FIG. 8, the display of the EGFRbinding domain as Fab (SI-1X2, SI-1X6) has stronger on-rate binding thantheir scFv forms (SI-1X1, SI-1X5, respectively). Here, both EGFR andHer3 exhibit the same Fab»scFv on-rate trend. SI-1X3 and SI-1X4 do notexhibit binding to monomeric EGFR, however each has the ability to bindHer3, as expected since each molecule uses the same αHer3 binding domainas SI-1X1, SI-1X2, SI-1X5, and SI-1X6. SI-1X3 and SI-1X4 areinvestigated for dimeric EGFR binding in an ELISA below.

Example 4: Dimeric EGFR ELISA Assay

As observed earlier, SI-1X3 and SI-1X4 were unable to bind a monomericform of EGFR in a BLI assay (FIG. 9). It has been suggested that inorder for the αEGFR binding domain used in SI-105, SI-1X3, and SI-1X4 tobind to EGFR in vitro, bivalent binding is required (Perez et al, ChinClin Oncol 2014; 3(1):5). To observe this, we utilized ELISA forantibody binding relative to other EGFR binding antibodies using adimeric form of EGFR.

ELISA was performed using dimeric EGFR ECD reagent, SI-2C1, fused torabbit Fc created in house. EGFR was coated onto Maxisorp immunoplates(Nunc) at 3 μg/mL in PBS at 4° C. overnight. Plates were blocked in PBSwith 3% BSA and 0.05% Tween20 for 2 hours at room temperature.Antibodies were captured at starting at 10 ug/mL except for SI-105,SI-1X3, and SI-1X4 which started at 50 μg/mL for (reported in nM), allwith 3× dilutions in PBST (1% BSA) for 1 hour at room temperature. Goatαhuman IgG-HRP antibody (Jackson ImmunoResearch, 109-035-098) was usedfor detection of the Fc portion of the antibodies at 1:2000 dilution inPBST (1% BSA) and developed in TMB (Thermo Scientific) for 5 minuteswith 2M H₂SO₄ as a stop solution. 3 washes with PBST (1% BSA) wereperformed between each step. All data points were performed intriplicate and collected at 450 nm (FIG. 10). SI-105, SI-1X3, and SI-1X4all bound to the dimeric EGFR ECD in this ELISA format at highconcentrations as compared to the other molecules.

Example 5: Binding Kinetics of 105.2 and 1X4.2 Using Octet

Kinetics determined using ForteBio Octet Red96 instrument withanti-human Fc sensors (ForteBio, AHC #18-5060). Binding experimentsperformed at 30° C. with 1000 RPM mixing. EGFR protein is extracellulardomain (Met 1-Ser 645) of human EGFR with a C-terminal polyhistidinetag. All samples diluted in 10X Kinetics Buffer (ForteBio #18-5032).105.2, 1X6 and 1X4.2 were loaded onto 8 sensors at 10 μg/ml each for 300seconds followed by a Baseline for 60 seconds in 10X Kinetics Buffer.Association with EGFR protein was performed for 300 seconds with eachsensor in a single concentration of EGFR protein (300, 100, 33.33,11.11, 3.705, 1.235, 0.4116 and 0 nM). Dissociation was then performedin 10X Kinetics Buffer for 900 seconds. A typical association anddissociation trace for 105.2 and 1X4.2 is shown in FIG. 11.

Data analysis was performed using ForteBio Data Analysis Software v9.0.Software curve-fitting was performed and the four most optimal curvefits for each 105.2 (TABLE 2), 1X4.2 (TABLE 3) and 1X6 (TABLE 4) wereused and averaged to determine KD, k(on) and k(dis). The average KD forSI-105.2 and SI-1X4.2 were 19.2 nM and 18.4 nM respectively. The averageKD for SI-1C6 was 3.04 nM 105.2 and 1X4.2 contained five amino acidchanges as compared to 105 and 1X4 as described in example 1. Thesechanges accounted for improved binding to EGFR ECD when compared to datagenerated for 105 and 1X4 in FIG. 10.

TABLE 2 Summary of KD, KON and KDIS for 1C5.2 EGFR KD KON KDIS (NM) (M)(1/MS) (1/S) SI-1C5.2 300 3.74E−08 4.61E+04 1.72E−03 SI-1C5.2 1002.23E−08 7.89E+04 1.76E−03 SI-1C5.2 33.3 9.94E−09 1.60E+05 1.59E−03SI-1C5.2 11.1 7.08E−09 2.12E+05 1.50E−03 AVERAGES 1.92E−08 1.24E+051.64E−03

TABLE 3 Summary of KD, KON and KDIS for 1X4.2 EGFR KD KON KDIS (NM) (M)(1/MS) (1/S) SI-1X4.2 300 3.69E−08 4.63E+04 1.71E−03 SI-1X4.2 1002.10E−08 7.88E+04 1.65E−03 SI-1X4.2 33.3 9.44E−09 1.58E+05 1.49E−03SI-1X4.2 11.1 6.19E−09 2.18E+05 1.35E−03 AVERAGES 1.84E−08 1.25E+051.55E−03

TABLE 4 Summary of KD, KON and KDIS for 1X6 EGFR KD KON KDIS (NM) (M)(1/MS) (1/S) SI-1C6 300 3.04E−09 4.11E+05 1.25E−03 SI-1C6 100 3.04E−094.11E+05 1.25E−03 SI-1C6 33.3 3.04E−09 4.11E+05 1.25E−03 SI-1C6 11.13.04E−09 4.11E+05 1.25E−03 AVERAGES 3.04E−09 4.11E+05 1.25E−03

Example 6: Binding Tests of Example Bispecific Antibodies to Tumor CellLines

The bispecific antibodies SI-1X1, SI-1X2, SI-1X3, SI-1X4, SI-1X5, andSI-1X6, as well as an isotype control were tested for binding to thetumor cell lines, A431 (epidermoid carcinoma, ATCC CRL-1555) and BxPC3(pancreatic adenocarcinoma, ATCC CRL-1687) by flow cytometry. Cells weregrown in RPMI-1640 medium containing 10% fetal bovine serum and wereharvested for analysis while in exponential growth phase. Aliquots of5×10⁶ cells were washed once in PBS, then resuspended in 250 μl ofPBS+1% bovine serum albumin (BSA) and incubated at 4° C. for 15 minutesto block membranes from non-specific binding. 250 μl of antibody,diluted to 10 μg/ml in PBS/1% BSA, was added to each sample for a finalantibody concentration of 5 μg/ml. Cells were incubated in primaryantibody for 1 hour at 4° C. with mixing. Cells were then washed twicewith 1 ml PBS/1% BSA and then resuspended in 500 μl of PE-conjugatedmouse-anti-human IgG-Fc and incubated at 4° C. with mixing for 45minutes. Samples were again washed twice with 1 ml PBS/1% BSA,resuspended in 300 ml PBS and analyzed using a FACScalibur flowcytometer. For each sample, 10000 events were collected in the FL-2channel. Histograms were generated using FCS Express software and SI-1Xhistograms were overlaid with histograms from the isotype controlstaining. All six bispecific antibodies displayed histogram shifts withrespect to control staining indicating cell binding. This data isdisplayed in FIG. 12 (A431 cell binding) and FIG. 13 (BxPC3 cellbinding).

Example 7: Characterization of SI-105.2 and SI-1X4.2 by Cell BindingAssays

The bispecific antibody, SI-1X4.2, monospecific antibodies, SI-105.2 andSI-1C1, as well as an isotype control were tested for binding to thetumor cell lines, A431 (epidermoid carcinoma, ATCC CRL-1555) (FIG. 14)and FaDu (hypopharyngeal squamous cell carcinoma, ATCC HTB-43) (FIG. 15)by flow cytometry. Cells were grown in RPMI-1640 medium containing 10%fetal bovine serum and were harvested for analysis while in exponentialgrowth phase. Cells were washed once in PBS, then resuspended in PBS+5%fetal bovine serum albumin (FBS) at a concentration of 5×10⁶ cells/mland incubated at 4° C. for 15 minutes to block membranes fromnon-specific binding. 100 μl aliquots of cells were added to 100 μlaliquots of antibody (also diluted in PBS+5% FBS) in a 96-well plate.Samples were incubated in primary antibody for 45 minutes on ice. Cellswere then washed twice with 200 μl of PBS+5% FBS and then resuspended in100 μl of PE-conjugated mouse-anti-human IgG-Fc and incubated on ice 30minutes. Samples were again washed twice with 200 μl of PBS+5% FBS,resuspended in 200 μl PBS and analyzed using a FACScalibur flowcytometer. For each sample, 10000 events were collected in the FL-2channel. Histograms were analyzed using FCS Express software and thegeometric mean fluorescence intensity (GMFI) was determined for eachdata set. EC50 binding values were determined by plotting the GMFIversus antibody concentration using Graphpad Prism software. Thebispecific antibody, SI-1X4.2 displayed similar binding profile as themonospecific anti-EGFR antibody, SI-105.2 with similar EC50 in both celllines. The other monospecific anti-Her3 antibody, SI-1C1 binds weakly tothe two cell lines probably due to low level of expression of Her3 onthe surface of the cells. 105.2 and 1X4.2 contained five amino acidchanges as compared to 105 and 1X4 as described in example 1. Thesechanges accounted for improved binding to target cells when compared tothe parental molecule, 1X4.

Example 8: Anti-Proliferative Effect of SI-1X Antibodies on Tumor CellLines

To assess the growth inhibitory potential of anti-Her3/EGFR bispecificantibodies, the effect on proliferation of A431 cells (ATCC CRL-1555,Manassas, Va.) which are an epidermoid carcinoma tumor line was tested.The effect on proliferation of BxPC3 (ATCC CRL-1687, Manassas, Va.), apancreatic adenocarcinoma tumor line was also tested. For each line,cells were seeded into 96-well tissue culture plates at a density of6000 cells/well in 100 μl RPMI-1640 medium containing 1% fetal bovineserum. After 4 hours, test antibodies were added at variousconcentrations, ranging from 0.0015 nM to 100 nM. Cells were cultured inthe presence of test antibodies for 72 hours. To each well, 20 μl of MTSreagent (Promega, Madison, Wis.) was added and cells were incubated at37° C. for 2 hours. MTS is readily taken up by actively proliferatingcells, reduced into formazan (which readily absorbs light at 490 nm),and then secreted into the culture medium. Following incubation, OD490values were measured using a BioTek (Winooski, Vt.) ELx800 absorbancereader. OD490 values for control cells (treated with medium only) werealso obtained in this manner at the time of antibody addition in orderto establish baseline metabolic activity. Proliferation may becalculated by subtracting the control baseline OD490 from the 72 hourOD490. Data from antibody titrations was expressed at % of controlpopulation according to the following formula: % of controlproliferation=(test proliferation/control proliferation)*100.

The effects of various bispecific anti-Her3/anti-EGFR antibodies on A431cell proliferation are shown in FIG. 16 and FIG. 17. SI-1X2 demonstratedmore efficacious antiproliferative effect than the control antibodiesSI-1C1 (anti-Her3), SI-1C3 (anti-EGFR), or SI-1C1 and SI-1C3 appliedtogether. SI-1X1 exhibited antiproliferative effects, although not tothe degree seen with SI-1C3 and the combination of SI-1C1 and SI-1C3.Inhibition plots as well as IC50 values are shown in FIG. 17. Similarresults were observed for SI-1X5 and SI-1X6, where SI-1X6 is more potentthan SI-1X5 and the control antibody SI-1C1 (anti-Her3), however itdisplayed similar antiproliferative potential as the control antibodySI-1C6 (anti-EGFR) and the combination of SI-1C1 and SI-1C6. This may beseen along with IC50 values in FIG. 17.

These molecules were also tested for antiproliferative effects in theBxPC3 cell line (FIG. 18 and FIG. 19). Again, SI-1X2 demonstrated moreefficacious antiproliferative effect than the control antibodies SI-1C1(anti-Her3), SI-1C3 (anti-EGFR), or SI-1C1 and SI-1C3 applied together.SI-1X1 was more efficacious than SI-1C1, but weaker than SI-1C3 and thecombination of SI-1C1 and SI-1C3. Inhibition curves and IC50 values aredisplayed in FIG. 19. BxPC3 proliferation was more strongly inhibited byboth SI-1X5 and SI-1X6 than with the control antibodies SI-1C1(anti-Her3), SI-1C6 (anti-EGFR), or SI-1C1 and SI-1C6 in combination.This data along with IC50 values is shown in FIG. 19.

Example 9: Anti-Proliferative Effect of SI-105.2 and SI-1X4.2 on TumorCell Lines

To assess the growth inhibitory potential of anti-Her3/EGFR bispecificantibodies, the effect on proliferation of FaDu (nasopharyngeal squamouscell carcinoma line, ATCC HTB-43) and A431 (epidermoid carcinoma, ATCCCRL-1555) cells were tested. Cells were seeded into 96-well tissueculture plates at a density of 6000 cells/well in 100 μl RPMI-1640medium containing 1% fetal bovine serum. After 4 hours, test antibodieswere added at various concentrations, ranging from 0.0015 nM to 100 nM.Cells were cultured in the presence of test antibodies for 72 hours. Toeach well, 11 μl of alamar blue reagent (Thermo Scientific) was addedand cells were incubated at 37° C. for 2 hours. Alamar blue is readilytaken up by actively proliferating cells, reduced, and then secretedinto the culture medium. The reduced form of alamar blue is stronglyfluorescent. Following incubation, fluorescence was measured using aMolecular Devices (Sunnyvale, Calif.) FilterMax F5 multi-mode platereader using an excitation wavelength of 535 nm and an emissionwavelength of 595 nm. Fluorescence values for control cells (treatedwith medium only) were also obtained in this manner at the time ofantibody addition in order to establish baseline metabolic activity.Proliferation may be calculated by subtracting the control baselinefluorescence from the 72-hour fluorescence values. Data from antibodytitrations was expressed at % of control population according to thefollowing formula: % of control proliferation=(testproliferation/control proliferation)*100.

The effects of SI-105.2 and SI-1X4.2 on Fadu and A431 cell proliferationare shown in FIG. 20 and FIG. 21 respectively. In both cell lines,SI-1X4.2 demonstrated improved efficacious anti-proliferative effectthan the control antibodies, SI-105.2 (anti-EGFR Mab), SI-1C1 (anti-HeraMab) or SI-1C1 and SI-1C7 applied together.

Example 10: ADCC Activities of SI-1X Bispecific Antibodies

The ability of SI-1X antibodies to mediate cellular cytotoxicity againstseveral tumor cell lines was tested. Whole blood was obtained fromnormal, healthy volunteers. Blood was diluted with an equal volume ofphosphate buffered saline (PBS). 20 ml aliquots of diluted blood werecarefully layered over 15 ml Ficol Pacque PLUS (GE Life Sciences cat#17-1440-02; Pittsburgh, Pa.). Tubes were centrifuged at 300 g for 40minutes with no brake. Following centrifugation most of the plasma layerwas carefully aspirated and the buffy coat (containing PBMC) wascarefully removed with a pipet in the smallest possible volume. PBMCswere pooled in 50 ml tubes and PBS added to bring each tube up to 50 ml.Tubes were centrifuged at 1300 RPM for 10 minutes and the supernatantwas carefully aspirated. Cells were resuspended in 40 ml PBS andcentrifuged again. The process was repeated for a total of 2 washes.Following the final wash, cells were resuspended in 30 ml RPMI-1630+10%FBS and incubated overnight at 37° C., 5% CO₂.

Target cells tested were the head and neck squamous cell carcinoma line,FaDu (ATCC HTB-43, Manassas, Va.) and the non-small cell lungadenocarcinoma cell line, NCI-H1975 (ATCC CRL-5908, Manassas, Va.).Target cells were labeled with calcein as follows. Cells were grown asmonolayers and were detached by incubation with accutase. Cells werewashed twice in RPMI with no serum. 1 ml of cells at 4×10⁶ cells/ml wasmixed with 1 ml RPMI (no serum)+20 μM calcein AM (Sigma cat #C1359; St.Louis, Mo.). Cells were incubated at 37° C. for 30 minutes, with gentlemixing every 10 minutes. Following labeling, cells were washed twicewith 14 ml RPMI+10% FBS+2.5 mM probenecid (assay medium). Probenecid(Sigma cat #P8761; St. Louis, Mo.) is an anionic transporter inhibitorand is known to reduce spontaneous release of intracellular calcein.Cells were resuspended in 20 ml assay medium and allowed to recover for2 hours at 37° C., 5% CO₂. Cells were then washed once with assay mediumand diluted to 200,000 cells/ml. Aliquots of 50 μl (10,000 cells)calcein-labeled cells were aliquoted to 96-well round-bottom plates. 50μl of antibody (at 3X final concentration) was added to cells andallowed to bind for 40 minutes on ice. PBMCs from the previous day werecentrifuged at 300 g for 5 minutes, resuspended in 20 ml fresh assaymedium, counted, and diluted to 6×10⁶ cells/ml. 50 μl PBMC (300,000)were added to each well and plates incubated at 37° C., 5% CO₂ for 4hours. Each antibody was titrated in triplicate via 10-fold serialdilutions, starting at 50 nM and going down to 0.00005 nM. Control wellswere also set up containing labeled target cells in the absence ofantibody and effector cells in order to measure maximal and spontaneouscalcein release.

At the end of the 4-hour incubation, 50 μl of assay medium containing 8%IGEPAL CA-630 (Sigma cat #18896; St. Louis, Mo.) was added to controlwells containing labeled target cells only (to measure the maximalcalcein release). 50 μl of assay medium was added to all the other wellsto bring the total volume to 200 μl per well. Plates were centrifuged at2000 RPM for 10 minutes and 150 μl supernatant was carefully transferredto V-bottom 96-well plates. These plates were centrifuged at 2000 RPMfor an additional 10 minutes and 100 ml supernatant was carefullytransferred to black, clear-bottom 96-well plates. Calcein in thesupernatant was quantitated by measuring the fluorescence of each sampleusing an excitation wavelength of 485 nM and an emission wavelength of535 nM. The percentage of specific lysis was calculated as follows:

% specific lysis=[(test sample value−spontaneous release)/(maximalrelease−spontaneous release)]*100

The data is shown in FIG. 22 and FIG. 23. For both cell lines, SI-1X6.4mediated cellular cytotoxicity, but was not particularly more effectivethan the control antibodies, SI-1C6.2, SI-1C7, or the combination ofSI-1C6.2+SI-1C7. SI-1X6.4 did mediate cytotoxicity with a lower EC50than our benchmark antibody, SI-1C4. For both cell lines, SI-1X4.2mediated cellular cytotoxicity at about the same degree as the controlantibodies. However, it was not as effective as mediating cellularcytotoxicity as the benchmark, SI-1C4. This is likely due to the loweraffinity of SI-1X4.2.

Example 11: Thermal Stability of SI-1X Bispecific Antibodies

Protein Thermal Shift Study was performed for protein thermal stabilityanalysis. Protein melt reactions were set up using Protein Thermal ShiftBuffer™ and the Protein Thermal Shift Dye™ (Applied Biosystems). Inbrief, the 20 ul reaction mixture contains 5 ug protein, 5 ul ProteinThermal Shift Buffer™ and 2.5μ 8× diluted Protein Thermal Shift™ Dye.For the negative control, PBS was used instead. The reaction mixture wasadded into MicroAmp Optical Reaction Plate and sealed with MicroAmpOptical Adhesive Film. Each sample consisted of 4 repeats. The proteinmelt reactions were run on Applied Biosystem Real-Time PCR System from25-90° C. in 1% increment and then analyzed by Protein Thermal ShiftSoftware™. FIG. 24 shows the thermal curve of SI-1X2, SI-1X4.2,SI-1X6.4, SI-1C3, SI-1C3, SI-1C6.2, SI-105.2 and SI-1C7. TABLE 5 showsTm for these molecules. Tm is defined as the temperature needed tounfold 50% of the protein. The bispecific molecules, 1X2, 1X4.2 and 1X6all have Tm around 66° C. which are comparable to all the MAbs (1C3,1C6.2, 105.2) and the Fc-scFv (1C7) molecules.

TABLE 5 Protein Tm Name (° C.) SI-1X2 66.52 SI-1C3 70.06 SI-1X4.2 66.94SI-1C5.2 70.26 SI-1X6.4 66.50 SI-1C6.2 70.12 SI-1C7 66.40

Example 12: Serum Stability of SI-1X Bispecific Antibodies

Serum stability of the molecules SI-105.2, SI-1C6.2, SI-1X4.2, andSI-1X6.4 was determined by comparative binding to monomeric EGFR ECD byELISA after incubation at 100 μg/mL in 95% human serum (AtlantaBiologics, S40110) at 37° C. for Days 0, 3, and 7 time points with anextra time point of 55° C. on Day 7 to provide a known condition wheredegradation occurs. ELISA plates were coated with monomeric EGFR ECD(SI-2R4) at 3 μg/mL in PBS at 4° C. overnight. Coated ELISA plates wereblocked with 3% BSA PBST for 2 hours at 25° C. and then washed 3 timeswith PBST. SI-1C6.2 and SI-1X6.4 were diluted 1:10 with 1% BSA PBST anddiluted 4x across the plate. SI-105.2 and SI-1X4.2 were diluted 1:2 with1% BSA PBST and diluted 4× across the plate and incubated at 25° C. for1 hour. 3 more washes with PBST were performed before antigen capturewith 1 μg/mL Her3 ECD Rabbit IgG1 (SI-1R1) for 1 hour at 25° C. in 1%BSA PBST. 3 more washes with PBST were performed before goat anti-rabbitIgG-HRP (Bio-Rad 172-1019) secondary antibody was applied at 1:5000dilution in 1% BSA PBST at 25° C. for 1 hour. 3 final washes with PBSTbefore development with 100 μl Pierce 1-step Ultra TMB ELISA (Pierce,34028) for 10 minutes with a final quench of 100 μl 2M H₂SO₄. Plateswere read at 450 nm. ELISA data was plotted and curves created usingGraphPad Prism 6.

Results of the ELISA are reported by EC50 on FIG. 25 and indicate afavorable profile of minor degradation when held at 37° C. When placedin 55° C., the EC50 shifts roughly a log as the molecules are subjectedto degradation conditions. EC50 values for SI-105.2 shift from 589.7 pMon Day 0 to 755.2 pM on Day 7 at 37° C. (Δ165.5 pM) with a shift to6.522 nM on Day 7 at 55° C. (Δ5932.3 pM). EC50 values for SI-1C6 shiftfrom 218.2 pM on Day 0 to 226.6 pM on Day 7 at 37° C. (Δ8.4 pM) with ashift to 1.322 nM on Day 7 at 55° C. (Δ1103 pM). EC50 values forSI-1X4.2 shift from 429.3 pM on Day 0 to 466.7 pM on Day 7 at 37° C.(Δ37.4 pM) with a shift to 4.248 nM on Day 7 at 55° C. (Δ3818.7 pM).EC50 values for SI-1X6 shift from 209.3 pM on Day 0 to 237.3 pM on Day 7at 37° C. (Δ28 pM) with a shift to 4.112 nM on Day 7 at 55° C. (Δ3902.7pM).

Example 13: PK Half-Life of SI-1X Molecules

To test their half-life in vivo, pharmacokinetic experiments wereperformed in SD rats. A single, intravenous tail vein injection ofbispecific Abs (1C6 10 mg/kg, 1X6 10 mg/kg, 1X2 10 mg/kg, 1X4 32 mg/kg)were given to groups of 4 female rats randomized by body weight (190-212g range). Blood (˜150 μL) was drawn from the orbital plexus at each timepoint, processed for serum, and stored at −80° C. until analysis. Studydurations were 28 days.

Antibody concentrations were determined using three ELISA assays. Inassay 1 (EGFR ECD coated ELISA), recombinant EGFR-rabbit Fc was coatedto the plate, wells were washed with PBST (phosphate buffered salinewith 0.05% Tween) and blocked with 1% BSA in PBST. Serum or serumdiluted standards were then added, followed by PBST washing, addition ofHRP labeled rabbit-anti-human IgG (BOSTER), and additional PBST washing.TMB was then added and the plates were incubated 2.5 minutes in thedark. Color reaction was stopped by adding 2M sulfuric acid. Plate wasread at 450 nm wavelength. For assay 2 (Hera coated ELISA), serum wasdetected using a similar ELISA, but recombinant HER3-His was used ascapture reagent. For assay 3 (Sandwich ELISA), recombinant HER3-His wascoated, serum or serum diluted standard were added, followed by PBSTwashing, addition of EGFR-rabbit Fc in PBST, and additional PBSTwashing. HRP labeled goat-anti-rabbit IgG (BOSTER) was then added. PKparameters were determined with a non-compartmental model.

FIGS. 26-28 show serum concentration data for four antibodies with threedifferent assays respectively. Fitted PK parameters from in vivo PKstudies are provided in TABLE 6. PK data include half-life, whichrepresents the beta phase that characterizes elimination of antibodyfrom serum and Cmax, which represents the maximal observed serumconcentration, AUC, which represents the area under the concentrationtime curve.

TABLE 6 Half- Life Cmax AUC Assay Sample (h) (μg/ml) (μg/ml*h) EGFRSI-1X6 159 325.5 18250.6 Coated SI-1X2 130 280.3 18889.8 ELISA SI-1X4.2146 627.8 31317.0 SI-1C6 130 196.4  3790.3 Her3 SI-1X6 142 236.7 14213.6Coated SI-1X2 136 264.8 19012.2 ELISA SI-1X4.2 124 715.6 40063.4Sandwich SI-1X6 136 301.6 14182.6 ELISA SI-1X2 123 297.6 17203.9SI-1X4.2 211 518.9 34874.6

Example 14: Mouse Xenograft Studies

The example tested the activity of SI-1X2, SI-1X4.2 and SI-1X6 ofconcomitant blockade of EGFR, HER3 in preclinical models of Fadu (headand neck squamous cell carcinoma xenograft model) and compared theirpotency with cetuximab and cetuximab in combination with an anti-HER3antibody.

All mouse studies were conducted through Institutional Animal care andused committee-approved animal protocols in accordance withinstitutional guidelines. Six-week-old female Balb/c Nude mice werepurchased from Beijing Vital River Laboratories and housed inair-filtered laminar flow cabinets with a 12-hour light cycle and foodand water ad libitum. The size of the animal groups was calculated tomeasure means difference between placebo and treatment groups of 25%with a power of 80% and a P value of 0.01. Host mice carrying xenograftswere randomly and equally assigned to either control or treatmentgroups. Animal experiments were conducted in a controlled andnon-blinded manner. For cell line-derived xenograft studies, mice wereinjected subcutaneously with 2×1C6 Fadu suspended 150 μl of culturemedium per mouse.

Once tumors reached an average volume of 100-250 mm3, mice wererandomized into 9 groups, with 6 mice per group. Vehicle Control, 1C6(25 mg/kg), 1C4 (25 mg/kg), 1C6+1C1 (25 mg/kg+50 mg/kg), SI-1X2 (25mg/kg), SI-1X6 (10 mg/kg), SI-1X6 (25 mg/kg), and SI-1X4.2 (10 mg/kg)SI-1X4 (25 mg/kg). All test articles were administered once weekly viaintravenous injection. Tumors were measured by digital caliper over theentire treatment period every 3 days and the volume was determined usingthe following formula: ½×length×width2. The body weight of mice wererecorded before the first dose and followed by every week during thetreatment period and recovery period.

All the test groups of SI-1X2, SI-1X6 and SI-1X4.2 and SI-1X6combination yielded significantly tumor growth inhibition compared topositive control of SI-1C6 excluding the low dose SI-1X4.2 10 mg/kggroup (FIGS. 29-30). Moreover, no relapses were observed 2 weeks aftertreatment cessation excluding the low dose SI-1X4.2 10 mg/kg group.

Pharmaceutical Compositions

The term “effective amount” refers to an amount of a drug effective toachieve a desired effect, e.g., to ameliorate disease in a subject.Where the disease is a caner, the effective amount of the drug mayinhibit (for example, slow to some extent, inhibit or stop) one or moreof the following example characteristics including, without limitation,cancer cell growth, cancer cell proliferation, cancer cell motility,cancer cell infiltration into peripheral organs, tumor metastasis, andtumor growth. Wherein the disease is a caner, the effective amount ofthe drug may alternatively do one or more of the following whenadministered to a subject: slow or stop tumor growth, reduce tumor size(for example, volume or mass), relieve to some extent one or more of thesymptoms associated with the cancer, extend progression free survival,result in an objective response (including, for example, a partialresponse or a complete response), and increase overall survival time. Tothe extent the drug may prevent growth and/or kill existing cancercells, it is cytostatic and/or cytotoxic.

With respect to the formulation of suitable compositions foradministration to a subject such as a human patient in need oftreatment, the antibodies disclosed herein may be mixed or combined withpharmaceutically acceptable carriers known in the art dependent upon thechosen route of administration. There are no particular limitations tothe modes of application of the antibodies disclosed herein, and thechoice of suitable administration routes and suitable compositions areknown in the art without undue experimentation.

Although many forms of administration are possible, an exampleadministration form would be a solution for injection, in particular forintravenous or intra-arterial injection. Usually, a suitablepharmaceutical composition for injection may include pharmaceuticallysuitable carriers or excipients such as, without limitation, a buffer, asurfactant, or a stabilizer agent. Example buffers may include, withoutlimitation, acetate, phosphate or citrate buffer. Example surfactantsmay include, without limitation, polysorbate. Example stabilizer mayinclude, without limitation, human albumin.

Similarly, persons skilled in the art have the ability to determine theeffective amount or concentration of the antibodies disclosed therein toeffective treat a condition such as a cancer. Other parameters such asthe proportions of the various components in the pharmaceuticalcomposition, administration does and frequency may be obtained by personskilled in the art without undue experimentation. For example, asuitable solution for injection may contain, without limitation, fromabout 1 to about 20, from about 1 to about 10 mg antibodies per ml. Theexample dose may be, without limitation, from about 0.1 to about 20,from about 1 to about 5 mg/Kg body weight. The example administrationfrequency could be, without limitation, once per day or three times perweek.

While the present disclosure has been described with reference toparticular embodiments or examples, it may be understood that theembodiments are illustrative and that the disclosure scope is not solimited. Alternative embodiments of the present disclosure may becomeapparent to those having ordinary skill in the art to which the presentdisclosure pertains. Such alternate embodiments are considered to beencompassed within the scope of the present disclosure. Accordingly, thescope of the present disclosure is defined by the appended claims and issupported by the foregoing description.

What is claimed is:
 1. A bispecific tetravalent antibody, saidbispecific tetravalent antibody comprising: two IgG1 heavy chains; twokappa light chains; and two single chain Fv (scFv) domains; wherein thetwo IgG1 heavy chains and kappa light chains form an IgG moiety with abinding specificity to a first member of the EGFR family; wherein thetwo scFv domains have a binding specificity to a second member of theEGFR family, and each scFv domain is connected to the C-terminus ofeither of the IgG1 heavy chains by a connector with an amino acidsequence of (gly-gly-gly-gly-ser)_(n), to provide a IgG1-connectorconnection, wherein n is an integral of at least 1; and wherein eachscFv domain has a structure order of N terminus-variable heavychain-linker-variable light chain-C terminus or N-terminus-variablelight chain-linker-variable heavy chain-C-terminus, and wherein thelinker is comprised of amino acid sequence of (gly-gly-gly-gly-ser)_(m),wherein m is an integral of at least
 3. 2. The bispecific tetravalentantibody of claim 1, wherein n is an integral between 1 to
 10. 3. Thebispecific tetravalent antibody of claim 1, wherein m is 3, 4, 5, or 6.4. The bispecific tetravalent antibody of claim 1, wherein at least oneof the IgG1 heavy chains is a humanized or human IgG1 heavy chain. 5.The bispecific tetravalent antibody of claim 1, wherein both IgG1 heavychains are humanized or human IgG1 heavy chains.
 6. The bispecifictetravalent antibody of claim 1, wherein at least one of the kappa lightchains is a humanized or human kappa light chain.
 7. The bispecifictetravalent antibody of claim 1, wherein both kappa light chains arehumanized or human kappa light chains.
 8. The bispecific tetravalentantibody of claim 1, wherein the first or the second member of the EGFRfamily comprises HER3, EGFR, a fragment or a derivative thereof.
 9. Thebispecific tetravalent antibody of claim 1, wherein the first or thesecond member of the EGFR family is independently selected from a groupconsisting of HER3, EGFR, a fragment or a derivative thereof.
 10. Thebispecific tetravalent antibody of claim 1, wherein the IgG moiety has abinding specificity for HER3.
 11. The bispecific tetravalent antibody ofclaim 1, wherein the scFv domains have a binding specificity for EGFR.12. The bispecific tetravalent antibody of claim 1, wherein the IgGmoiety has a binding specificity for HER3 and the scFv domains have abinding specificity for EGFR simultaneously.
 13. The bispecifictetravalent antibody of claim 1, wherein the IgG moiety has a bindingspecificity for EGFR,
 14. The bispecific tetravalent antibody of claim1, wherein the scFv domains have a binding specificity for HER3.
 15. Thebispecific tetravalent antibody of claim 1, wherein the IgG moiety has abinding specificity for EGFR and the scFv domains have a bindingspecificity for HER3 simultaneously.
 16. The bispecific tetravalentantibody of claim 1, wherein the C terminus of at least one of the IgG1heavy chains misses an amino acid residue.
 17. The bispecifictetravalent antibody of claim 16, wherein the amino acid residue is alysine.
 18. The bispecific tetravalent antibody of claim 1, wherein theIgG1-connector connection is resistant to protease activity.
 19. Thebispecific tetravalent antibody of claim 1, wherein at least one of theIgG1 heavy chains comprises two mutations in the CH3 domain, and whereinthe two mutations are reversion to the common residues in human CH3domain.
 20. The bispecific tetravalent antibody of claim 1, wherein atleast one of the IgG1 heavy chains comprises an amino acid comprisingSEQ ID NO 7, 15, 23, 31, 39, 47, and
 127. 21. The bispecific tetravalentantibody of claim 1, wherein the IgG1 heavy chain, connector, and scFvdomain have an amino acid comprising SEQ ID NO 56, 66, 76, 86, 98, 108,118, and
 136. 22. The bispecific tetravalent antibody of claim 1,wherein at least one of the kappa light chains comprises an amino acidsequence comprising SEQ ID NO 3, 11, 19, 27, 35, 43, 51, 61, 71, 81, 92,103, 113, 123, and
 131. 23. The bispecific tetravalent antibody of claim1, wherein at least one of variable light chain comprises an amino acidsequence comprising SEQ ID NO 4, 12, 20, 28, 36, 44, 52, 62, 72, 82, 93,104, 114, 124, and
 132. 24. The bispecific tetravalent antibody of claim1, wherein at least one of variable heavy chain comprises an amino acidsequence comprising SEQ ID NO 8, 16, 24, 32, 40, 48, 57, 67, 77, 87, 99,109, 119, 128, and
 137. 25. The bispecific tetravalent antibody of claim1, wherein the IgG moiety has a binding specificity for HER3, and thescFv domains have a binding specificity for EGFR; wherein the IgG1 heavychain, connector, and scFv domain have an amino acid sequence of SEQ IDNO 56, and the kappa light chain has an amino acid sequence of SEQ ID NO51.
 26. The bispecific tetravalent antibody of claim 1, wherein the IgGmoiety has a binding specificity for HER3, and the scFv domains have abinding specificity for EGFR; wherein the IgG1 heavy chain, connector,and scFv domain have an amino acid sequence of SEQ ID NO 76, and thekappa light chain has an amino acid sequence of SEQ ID NO
 71. 27. Thebispecific tetravalent antibody of claim 1, wherein the IgG moiety has abinding specificity for HER3, and the scFv domains have a bindingspecificity for EGFR; wherein the IgG1 heavy chain, connector, and scFvdomain have an amino acid sequence of SEQ ID NO 108, and the kappa lightchain has an amino acid sequence of SEQ ID NO
 103. 28. The bispecifictetravalent antibody of claim 1, wherein the IgG moiety has a bindingspecificity for EGFR, and the scFv domains have a binding specificityfor HER3; wherein the IgG1 heavy chain, connector, and scFv domain havean amino acid sequence of SEQ ID NO 66, and the kappa light chain has anamino acid sequence of SEQ ID NO
 61. 29. The bispecific tetravalentantibody of claim 1, wherein the IgG moiety has a binding specificityfor EGFR, and the scFv domains have a binding specificity for HER3;wherein the IgG1 heavy chain, connector, and scFv domain have an aminoacid sequence of SEQ ID NO 86, and the kappa light chain has an aminoacid sequence of SEQ ID NO
 81. 30. The bispecific tetravalent antibodyof claim 1, wherein the IgG moiety has a binding specificity for EGFR,and the scFv domains have a binding specificity for HER3; wherein theIgG1 heavy chain, connector, and scFv domain have an amino acid sequenceof SEQ ID NO 98, and the kappa light chain has an amino acid sequence ofSEQ ID NO
 92. 31. The bispecific tetravalent antibody of claim 1,wherein the IgG moiety has a binding specificity for EGFR, and the scFvdomains have a binding specificity for HER3; wherein the IgG1 heavychain, connector, and scFv domain have an amino acid sequence of SEQ IDNO 118, and the kappa light chain has an amino acid sequence of SEQ IDNO
 113. 32. The bispecific tetravalent antibody of claim 1, wherein theIgG moiety has a binding specificity for EGFR, and the scFv domains havea binding specificity for HER3; wherein the IgG1 heavy chain, connector,and scFv domain have an amino acid sequence of SEQ ID NO 136, and thekappa light chain has an amino acid sequence of SEQ ID NO
 131. 33. Thebispecific tetravalent antibody of claim 1, wherein the antibodyinhibits cancer cell growth.
 34. The bispecific tetravalent antibody ofclaim 1, wherein the antibody binds to EGRF and HER3 with a Kd less than50 nM.
 35. The bispecific tetravalent antibody of claim 1, wherein theantibody simultaneously binds to EGRF with a Kd less than 50 nM andbinds to HER3 with a Kd less than 50 nM.
 36. An IgG1 heavy chains forthe bispecific tetravalent antibody of claim 1, comprising an amino acidsequences selected from SEQ ID NO 7, 15, 23, 31, 39, 47, and 127
 37. Akappa light chain for the bispecific tetravalent antibody of claim 1,comprising an amino acid sequence selected from SEQ ID NO 3, 11, 19, 27,35, 43, 51, 61, 71, 81, 92, 103, 113, 123, and
 131. 38. A variable lightchain for the bispecific tetravalent antibody of claim 1, comprising anamino acid sequence selected from SEQ ID NO 4, 12, 20, 28, 36, 44, 52,62, 72, 82, 93, 104, 114, 124, and 132
 39. A variable heavy chain forthe bispecific tetravalent antibody of claim 1, comprising an amino acidsequence selected from SEQ ID NO 8, 16, 24, 32, 40, 48, 57, 67, 77, 87,99, 109, 119, 128, and
 137. 40. An isolated nucleic acid encoding theantibody of claim 1, the IgG1 heavy Chain of claim 36, the kappa lightchain of claim 37, the variable light chain of claim 38, or the variableheavy chain of claim
 39. 41. An expression vector comprising theisolated nucleic acid of claim claim
 40. 42. The expression vector ofclaim 41, wherein the vector is expressible in a cell.
 43. A host cellcomprising the nucleic acid of claim
 40. 44. A host cell comprising theexpression vector of claim
 43. 45. The host cell of claim 44, whereinthe host cell is a prokaryotic cell or a eukaryotic cell.
 46. A methodof producing an antibody comprising culturing the host cell of one ofclaims 43-45 so that the antibody is produced.
 47. An immunoconjugatecomprising the antibody of claim 1 and a cytotoxic agent.
 48. Apharmaceutical composition, comprising the bispecific tetravalentantibody of claim 1 and a pharmaceutically acceptable carrier.
 49. Thepharmaceutical composition of claim 48, further comprising radioisotope,radionuclide, a toxin, a therapeutic agent, a chemotherapeutic agent ora combination thereof.
 50. A pharmaceutical composition, comprising theimmunoconjugate of claim 47 and a pharmaceutically acceptable carrier.51. A method of treating a subject with a cancer, comprisingadministering to the subject an effective amount of the bispecifictetravalent antibody of claim
 1. 52. The method of claim 51, wherein thecancer comprises cells expressing at least two members of EGFR family.53. The method of claim 51, wherein the cancer comprises breast cancer,colorectal cancer, pancreatic cancer, head and neck cancer, melanoma,ovarian cancer, prostate cancer, non-small lung cell cancer, glioma,esophageal cancer, nasopharyngeal cancer, anal cancer, rectal cancer,gastric cancer, bladder cancer, cervical cancer, or brain cancer. 54.The method of claim 51, further comprising co-administering an effectiveamount of a therapeutic agent.
 55. The method of claim 54, wherein thetherapeutic agent comprises an antibody, a chemotherapy agent, anenzyme, or a combination thereof.
 56. The method of claim 54, whereinthe therapeutic agent comprises an anti-estrogen agent, a receptortyrosine inhibitor, or a combination thereof.
 57. The method of claim54, wherein the therapeutic agent comprises capecitabine, cisplatin,trastuzumab, fulvestrant, tamoxifen, letrozole, exemestane, anastrozole,aminoglutethimide, testolactone, vorozole, formestane, fadrozole,letrozole, erlotinib, lafatinib, dasatinib, gefitinib, imatinib,pazopinib, lapatinib, sunitinib, nilotinib, sorafenib, nab-palitaxel, aderivative or a combination thereof.
 58. The method of claim 54, whereinthe therapeutic agent comprises a check point inhibitor.
 59. The methodof claim 54, wherein the therapeutic agent comprises PD1, PDL1, CTLA4,4-1BB, OX40, GITR, TIM3, LAG3, TIGIT, CD40, CD27, HVEM, BTLA, VISTA,B7H4, a derivative or a combination thereof.
 60. The method of claim 51,wherein the subject is a human.
 61. A method of inhibiting a biologicalactivity of a HER receptor in a subject, comprising administering to thesubject an effective amount of the antibody of claim 1 to inhibit abiological activity of a HER receptor.
 62. A solution comprising aneffective concentration of the bispecific tetravalent antibody of claim1, wherein the solution is blood plasma in a subject.